Biology subjects

Maniatis, T.

Publications and source records attributed to Maniatis, T..

8 recordsLinked to original sources

bioRxiv2025

Enhancer Dynamics and Spatial Organization Drive Anatomically Restricted Cellular States in the Human Spinal Cord

Here, we report the spatial organization of RNA transcription and associated enhancer dynamics in the human spinal cord at single-cell and single-molecule resolution. We expand traditional multiomic measurements to reveal epigenetically poised and bivalent active transcriptional enhancer states that define cell type specification. Simultaneous detection of chromatin accessibility and histone modifications in spinal cord nuclei reveals previously unobserved cell-type specific cryptic enhancer activity, in which transcriptional activation is uncoupled from chromatin accessibility. Such cryptic enhancers define both stable cell type identity and transitions between cells undergoing differentiation. We also define glial cell gene regulatory networks that reorganize along the rostrocaudal axis, revealing anatomical differences in gene regulation. Finally, we identify the spatial organization of cells into distinct cellular organizations and address the functional significance of this observation in the context of paracrine signaling. We conclude that cellular diversity is best captured through the lens of enhancer state and intercellular interactions that drive transitions in cellular state. This study provides fundamental insights into the cellular organization of the healthy human spinal cord.

neuroscience↗

bioRxiv2022

Complex Intracellular Mechanisms of TBK1 Kinase Activation Revealed by a Specific Small Molecule Inhibitor

TANK-binding kinase 1 (TBK1), a dimeric serine/threonine protein kinase, plays a critical role in multiple signaling pathways including innate immunity, autophagy and cell death. TBK1 is activated by the phosphorylation of an essential serine residue 172 (S172) within the activation loop of the kinase domain, and this phosphorylation can occur by trans-autophosphorylation: one TBK1 dimer phosphorylates a second dimer at S172. Here we show that phosphorylation of TBK1 S172 in cultured human cells in response to multiple inducers is reduced, but not abolished by the highly specific and potent TBK1 small molecule inhibitor GSK8612. Thus, upstream kinase(s) must phosphorylate TBK1 in response to inducers in cultured cells. We show that distinct upstream kinases are recruited for the activation of TBK1 in response to different inducers. We also identify extensive crosstalk among TBK1, IKK{beta} and IKK{varepsilon} kinases in the cellular response to various inducers. In addition, we show that dsDNA and dsRNA trigger dynamic intracellular translocation of TBK1, leading to its localization and activation on the Golgi apparatus or mitochondria, respectively. GSK8612 does not block the intracellular localization of TBK1. We conclude that TBK1 is activated by both upstream kinase phosphorylation and by trans-autophosphorylation, and the signal-dependent spatial engagement of TBK1 with other signaling molecules is a fundamental mechanism for its specific activation by multiple inducers.

molecular biology↗

bioRxiv2022

γ-Protocadherins control synapse formation and peripheral branching of touch sensory neurons

Light touch sensation begins with activation of low-threshold mechanoreceptor (LTMR) endings in the skin and propagation of their signals to the spinal cord and brainstem. We found that the clustered protocadherin gamma (Pcdhg) gene locus, which encodes 22 cell-surface homophilic binding proteins, is required in somatosensory neurons for normal behavioral reactivity to a range of tactile stimuli. Developmentally, distinct Pcdhg isoforms mediate LTMR synapse formation through neuron-neuron interactions and peripheral axonal branching through neuron-glia interactions. The Pcdhgc3 isoform mediates homophilic interactions between sensory axons and spinal cord neurons to promote synapse formation in vivo, and is sufficient to induce postsynaptic specializations in vitro. Moreover, loss of Pcdhgs and somatosensory synaptic inputs to the dorsal horn lead to fewer corticospinal synapses onto dorsal horn neurons. These findings reveal essential roles for Pcdhg isoform diversity in somatosensory neuron synapse formation, peripheral axonal branching, and step-wise assembly of central mechanosensory circuitry.

neuroscience↗

bioRxiv2021

ALS and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy

TANK-binding kinase 1 (TBK1) is a multi-functional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD), including missense mutations that disrupt the ability of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), auto-activate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presense of either WT- or kinase inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals. SIGNIFICANCE STATEMENTMissense mutations in TANK-binding kinase 1 (TBK1) have various biophysical and biochemical effects on the molecule, and are associated with the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). TBK1 plays an essential role in clearing damaged mitochondria. Here, we investigate the impact of 10 ALS-linked TBK1 mutations on the critical early stage of mitophagy. We find that both TBK1 recruitment and kinase activity contribute to the clearance of the damaged mitochondria. Furthermore, in neurons, expression of TBK1 mutants alone affects mitochondrial network health. Our investigation utilizes disease-linked mutations to further refine the current model of mitophagy, identifying crosstalk between the regulatory kinases TBK1 and ULK1, and providing new insights into the roles of TBK1 in neurodegenerative pathogenesis.

cell biology↗

bioRxiv2020

Clustered γ-Protocadherins Regulate Cortical Interneuron Programmed Cell Death.

Cortical function critically depends on inhibitory/excitatory balance. GABAergic cortical inhibitory interneurons (cINs) are born in the ventral forebrain. After completing their migration into cortex, their final numbers are adjusted-during a period of postnatal development - by programmed cell death (PCD). The mechanisms that regulate cIN elimination remain controversial. Here we show that genes in the protocadherin (Pcdh)-{gamma} gene cluster, but not in the Pcdh- or Pcdh-{beta} clusters, are required for survival of cINs through a BAX-dependent mechanism. Surprisingly, the physiological and morphological properties of Pcdh-{gamma} deficient and wild type cINs during PCD were indistinguishable. Co-transplantation of wild type and Pcdh-{gamma} deficient interneuron precursor cells demonstrate that: 1) the number of mutant cINs eliminated was much higher than that of wild type cells, but the proportion of mutant or WT cells undergoing cell death was not affected by their density; 2) the presence of mutant cINs increases cell death among wild-type counterparts, and 3) cIN survival is dependent on the expression of Pcdh-{gamma} C3, C4, and C5. We conclude that Pcdh-{gamma}, and specifically {gamma}C3, {gamma}C4, and {gamma}C5, play a critical role in regulating cIN survival during the endogenous period of PCD. SignificanceGABAergic cortical inhibitory interneurons (cINs) in the cerebral cortex originate from the ventral embryonic forebrain. After a long migration, they come together with local excitatory neurons to form cortical circuits. These circuits are responsible for higher brain functions, and the improper balance of excitation/inhibition in the cortex can result in mental diseases. Therefore, an understanding of how the final number of cINs is determined is both biologically and, likely, therapeutically significant. Here we show that cell surface homophilic binding proteins belonging to the clustered protocadherin gene family, specifically three isoforms in the Pcdh-{gamma} cluster, play a key role in the regulation cIN programmed cell death. Co-transplantation of mutant and wild-type cINs shows that Pcdh-{gamma} genes have cell-autonomous and non-cell autonomous roles in the regulation of cIN cell death. This work will help identify the molecular mechanisms and cell-cell interactions that determine how the proper ratio of excitatory to inhibitory neurons is determined in the cerebral cortex.

neuroscience↗

bioRxiv2018

Regional collapsing of rare variation implicates specific genic regions in ALS

Large-scale sequencing efforts in amyotrophic lateral sclerosis (ALS) have implicated novel genes using gene-based collapsing methods. However, pathogenic mutations may be concentrated in specific genic regions. To address this, we developed two collapsing strategies, one focuses rare variation collapsing on homology-based protein domains as the unit for collapsing and another gene-level approach that, unlike standard methods, leverages existing evidence of purifying selection against missense variation on said domains. The application of these two collapsing methods to 3,093 ALS cases and 8,186 controls of European ancestry, and also 3,239 cases and 11,808 controls of diversified populations, pinpoints risk regions of ALS genes including SOD1, NEK1, TARDBP and FUS. While not clearly implicating novel ALS genes, the new analyses not only pinpoint risk regions in known genes but also highlight candidate genes as well.

genetics↗

bioRxiv2018

Antisense lncRNA transcription drives stochastic Protocadherin α promoter choice

Stochastic and combinatorial activation of clustered Protocadherin (Pcdh) , {beta}, and {gamma} gene promoters generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here we show that Pcdh promoter choice requires transcription of a long noncoding RNA (lncRNA) initiated from newly identified promoters located in the protein coding sequence of each Pcdh exon. Antisense transcription of the lncRNA through the sense promoter results in its activation and in DNA demethylation of the binding sites for the CCCTC-binding protein, CTCF, located in close proximity to both sense and antisense promoters. Increased CTCF binding promotes the assembly of long-range DNA contacts between the activated promoter and a neuron-specific enhancer, thus locking in the epigenetic state of the stochastically chosen Pcdh promoter. Examination of this hierarchical molecular mechanism in differentiating olfactory sensory neurons, suggests that antisense Pcdh transcription is a key prerequisite for stochastic Pcdh promoter choice in vivo.

genomics↗

bioRxiv2018

Cell type-specific CLIP reveals that NOVA regulates cytoskeleton interactions in motoneurons

BackgroundAlternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system (CNS). This is believed to involve differential regulation of RNA processing in various cell types. However, in vivo study of cell-type specific post-transcriptional regulation has been a challenge. Here, we developed a sensitive and stringent method combining genetics and CLIP (crosslinking and immunoprecipitation) to globally identify regulatory interactions between NOVA and RNA in the mouse spinal cord motoneurons (MNs).\n\nResultsWe developed a means of undertaking MN-specific CLIP to explore MN-specific protein-RNA interactions relative to studies of the whole spinal cord. This allowed us to pinpoint differential RNA regulation specific to MNs, revealing major role for NOVA in regulating cytoskeleton interactions in MNs. In particular, NOVA specifically promotes the palmitoylated isoform of a cytoskeleton protein Septin 8 in MNs, which enhances dendritic arborization.\n\nConclusionsOur study demonstrates that cell type-specific RNA regulation is important for fine-tuning motoneuron physiology, and highlights the value of defining RNA processing regulation at single cell type resolution.

neuroscience↗